Ubiquitin is a small 76-amino acid protein that is the founding member of a family of posttranslational modifiers known as the ubiquitin-like proteins (Ubls). FAT10, originally identified as diubiquitin, is composed of two ubiquitin-like domains and is also a member of the Ubl family. W. Fan et al., Immunogenetics, 1996, 44(2), 97-103. Ubls play key roles in controlling many biological processes including cell division, cell signaling and the immune response. Ubls are small proteins that are covalently attached to a lysine on a target protein via an isopeptide linkage with a C-terminal glycine of the Ubl. The Ubl molecule alters the molecular surface of the target protein and can affect such properties as protein-protein interactions, enzymatic activity, stability and cellular localization of the target.
There are 8 known human Ubl activating enzymes (known as E1s). B. A. Schulman and J. W. Harper, Nat Rev Mol Cell Biol, 2009, 10, 319-31. Ubiquitin and other Ubls are activated by specific E1 enzymes which catalyze the formation of an acyl-adenylate intermediate with the C-terminal glycine of the Ubl. The activated Ubl molecule is then transferred to the catalytic cysteine residue within the E1 enzyme through formation of a thioester intermediate. The E1-Ubl intermediate and an E2 interact, resulting in a thioester exchange wherein the Ubl is transferred to the active site cysteine of the E2. The Ubl is then conjugated to the target protein, either directly or in conjunction with an E3 ligase, through isopeptide bond formation with the amino group of a lysine side chain in the target protein. Eukaryotic cells possess ˜35 ubiquitin E2 enzymes and >500 ubiquitin E3 enzymes. The E3 enzymes are the specificity factors of the ubiquitin pathway which mediate the selective targeting of specific cellular substrate proteins. R. J. Deshaies and C. A. Joazeiro, Annu Rev Biochem, 2009, 78, 399-434; S. Lipkowitz and A. M. Weissman, Nat Rev Cancer, 2011, 11, 629-43; D. Rotin and S. Kumar, Nat Rev Mol Cell Biol, 2009, 10, 398-409.
Uba6 is a member of the E1 activating enzyme class of enzymes and was identified in 2007 as an alternate ubiquitin activating enzyme. J. Jin et al., Nature, 2007, 447(7148), 1135-38; Y. H. Chiu et al., Mol Cell, 2007, 27(6), 1014-23; C. Pelzer et al., J Biol Chem, 2007, 282(32), 23010-14. Uba6 has been shown to play a role in cytoplasmic N-end rule ubiquitin-mediated protein degradation. P. C. Lee et al., Mol Cell, 2011, 43(3), 392-405. The mouse knockout of Uba6 has an early embryonic lethal phenotype which supports an essential role for this enzyme. In addition tissue specific knockouts of Uba6 have unique phenotypes. Mice with Uba6 knocked out in all tissue originating from the neural crest have altered neuronal development. P. Lee, et al., Mol Cell, 2013, 50(2), 172-84. Uba6 also activates a Ubiquitin-like protein (Ubl) called FAT10 (or UBD). Y. H. Chiu et al., Mol Cell, 2007, 27(6), 1014-23. While this Ubl is dispensable for mouse development, the loss of FAT10 systemically is associated with a dramatic increase in sensitivity to bacterial challenge. A. Canaan, et al., Mol Cell Biol, 2006, 13:5180-89. Moreover, accumulation of FAT10 has been noted in hepatic and colon cancer as well as inflammatory bowel disease, Celiac disease and Crohn's disease. C. G. Lee, et al., Oncogene, 2003, 22(17), 2592-603; S. Lukasiak et al., Oncogene, 2008, 27(46), 6068-74. FAT10 expression has been shown to be negatively affected by the known tumor suppressor p53 (D. Zhang et al., Oncogene, 2006, 25(16), 2318-27) and in turn FAT10 conjugation has been shown to affect p53 function (T. Li, et al., Arch Biochem Biophys 2011, 509(2), 164-9). It has also been reported that FAT10 plays a role in mitotic progression, however this may only occur in certain cell types or conditions such as inflammation. Y. Merbl, Cell, 2013, 152, 1160-72. There is a Uba6-specific E2 enzyme (Use1) that has been identified to productively interact only with Uba6, but not Uba1. J. Jin et al., Nature, 2007, 447(7148), 1135-38. Use1 can accept activated ubiquitin or FAT10. A. Aichem et al., Nat Commun, 2010, 1, 13.
Although the role of Uba6 in ubiquitin metabolism may be modest relative to the better characterized Uba1, the knock out data confirms that Uba6 must play an essential role in cell proliferation. The essential function of Uba6 may not be confined to its activation of either ubiquitin or FAT10, but potentially a combination of the two, therefore representing a possible synthetic lethal interaction with the potential for anti-cancer effects. Accordingly, small molecule inhibitors of Uba6 would be expected to act as potent anti-proliferative agents such as in tumors that overexpress FAT10 (gastrointestinal, gynecological and hepatic origin), and may have utility as a general oncology chemotherapeutic L. Liu, Oncogene, 2013 advanced online publication Jul. 1, 2013. The inhibition of Uba6 function should also impact FAT10 metabolism. A small molecule inhibitor of Uba6 would therefore also be expected to have use in the anti-inflammatory and immunologic setting, such as in the treatment of inflammatory bowel disease, Crohn's disease (B. Frank et al., Int J Cancer, 2010, 927(12), 2822-30) and Celiac disease (A. Castellanos-Rubio et al., Hum Immunol, 2010, 79(1), 96-99).